Process for producing highly unsaturated fatty acid-containing lipid

ABSTRACT

The present invention provides a process for producing a highly unsaturated fatty acid-containing lipid which is less expensive than existing ones, which comprises culturing a microorganism belonging to the genus  Mortierella  with the use of, as a medium carbon source, a saccharified starch, which is less expensive than glucose, does not contribute to an increase in osmotic pressure of the culture medium, and can be utilized by the  Mortierella  microorganism, and collecting a highly unsaturated fatty acid-containing lipid from the culture.

TECHNICAL FIELD

The present invention relates to a process for producing a highlyunsaturated fatty acid-containing lipid, comprising culturing amicroorganism belonging to the genus Mortierella producing a highlyunsaturated fatty acid (hereinafter, referred to as a Mortierellamicroorganism) with the use of a saccharified starch as a carbon sourceof the culture medium, and collecting a highly unsaturated fattyacid-containing lipid from the culture.

BACKGROUND TECHNIQUE

In the present specification, an unsaturated fatty acid refers to afatty acid having one or more double bond(s) in a carbon chain and,among this, the fatty acid having a carbon number of 18 or more and twoor more double bonds is generally referred to as “highly unsaturatedfatty acid”. Examples of the highly unsaturated fatty acid includeγ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,eicosapentaenoic acid, mead acid, 6,9-octadecadienoic acid,8,11-eicosadienoic acid, etc. Most of the highly unsaturated fatty acidsgenerated by a Mortierella microorganism are produced as fatty acidsconstituting triglyceride and, besides this, also as fatty acidsconstituting various lipids such as diglyceride, monoglyceride, freefatty acid, phospholipid, etc. As used herein, the highly unsaturatedacid-containing lipid is a mixture of lipids containing these varioushighly unsaturated fatty acids, and an amount of arachidonic acidproduction is a value obtained by converting the amount of arachidonicacid existing as fatty acids constituting those various lipids into theamount of free fatty acids. In addition, a mixture of oligoglucose andglucose, which is obtained by treating a starch with a saccharifyingenzyme such as amylase, is referred to as “saccharified starch”, a sugardegrading enzyme such as amylase which is used for producing asaccharified starch is referred to as “saccharifying enzyme”, andtreatment of a starch with the enzyme is referred to as“saccharification”. A degradation degree of starch by saccharificationis referred to as “saccharification degree”, and the saccharificationdegree is expressed by the ratio of reducing sugar/total sugar (%) ofthe saccharified starch. For example, a ratio of reducing sugar/totalsugar of 50% indicates that the average chain length of α-glucan in thesaccharified starch is 2.

Highly unsaturated fatty acids such as arachidonic acid, along with DHA(docosahexaenoic acid), have drawn attention from a nutritional point ofview, especially as a component necessary for the growth of a baby.Lanting et al. conducted a 9-year follow-up study of babies who had beenbreastfed and babies who had been powdered formula-fed for three weeksor longer after birth. They examined the incidence of a minorneurological dysfunction from behavioral aspects and, as a result,reported that the incidence of neurological dysfunction in children whowere powdered formula-fed was 2-fold that of children who were breastfed(LANCET, vol.344, 1319-1322 (1994)). According to the report, the resultis due to the fact that highly unsaturated fatty acids such as DHA,arachidonic acid, etc., which are present in breast milk but are hardlypresent in powdered formula, are involved in the development of a baby'sbrain. Besides, results that highly unsaturated fatty acids are involvedin the development of brain and retina of a neonate, have beenfrequently reported (Carlson et al., Broc. Natl. Acad. Sci.90:1073-1077(1993)), and the importance of those highly unsaturatedfatty acids is drawing attention from a nutritional viewpoint for apremature baby and a neonate.

Highly unsaturated fatty acids are widely distributed in the livingworld and, for example, arachidonic acid has been separated from a lipidextracted from animal adrenal gland or liver. However, since a contentof the highly unsaturated fatty acid in animal organs is small, theextraction and separation of highly unsaturated fatty acids from ananimal organ is not a sufficient method for supplying a large amount ofhighly unsaturated fatty acids. For this reason, methods of obtaininghighly unsaturated fatty acids by culturing various microorganisms havebeen developed. Among the microorganisms, a Mortierella microorganism isknown as a microorganism producing a highly unsaturated fattyacid-containing lipid such as arachidonic acid, dihomo-y-linolenic acid,eicosapentaenoic acid, etc., and a process for producing a highlyunsaturated fatty acid-containing lipid by fermentation using thismicroorganism has been developed (JP-A No. 63-44891, JP-A No. 63-12290,JP-A No. 63-14696, JP-A No. 63-14697). Further, a process for producinga mead acid using a mutant in which Δ12 desaturation enzyme activitiesof a Mortierella microorganism are reduced or lost has also been known(JP-A No. 5-91888). In addition, a process for producingdihomo-γ-linolenic acid using a mutant in which Δ5 desaturation enzymeactivities are reduced or lost, which is obtained by subjecting aMortierella microorganism to mutation induction, has also been known(JP-A No. 5-91887).

Although it has already been known that a Mortierella microorganism hasthe ability to assimilate a starch, since its starch-assimilatingability is inferior as compared to the glucose-utilizing ability,glucose has been widely used as a medium carbon source for suchmicroorganism( Shinmenet al., Appl. Microbiol. Biotechnol. 31:11-16(1989), Aki et al., JAOCS 78:599-604 (2001)). As a result of payingattention to the starch-utilizing ability of a Mortierellamicroorganism, it has been reported that a secreted starch-degradingenzyme is isolated and purified, and the starch-degrading enzyme isidentified to be α-glucosidase (Tanaka et al., Bulletin of Japan Societyof Bioscience, Biotechnology, and Agrochemistry, March 1999). However,α-glucosidase, which is an exo-type amylase having a degradation patternof cleavage of glucose units from a non-reducing end of α-glucan, has alow activity to degrade high-molecular α-glucan, a representative ofwhich is a starch, and has a high activity of degrading an oligoglucosecomposed of several glucoses. This is thought to be one of the reasonswhy the starch-utilizing ability of a Mortierella microorganism is lowerthan the glucose-utilizing ability. On the other hand, when glucose isused as a carbon source, since an increase in osmotic pressure due tohigh concentration of glucose in the culture medium has adverseinfluence on the growth of cells of, and the productivity of a highlyunsaturated fatty acid-containing lipid of a Mortierella microorganism,there has been widely used a method comprising culturing themicroorganism with low concentration glucose and sequentially addingglucose during culture to compensate the utilized glucose (Shinmen etal., Appl. Microbiol. Biotechnol. 31:11-16(1989)). In addition, anattempt to produce a highly unsaturated fatty acid-containing lipidusing a Mortierella microorganism having resistance to highconcentration glucose (International Publication No. WO98/39468) hasalso been tried. A carbon source of the culture medium accounts for themajority of raw material costs and, if this can be changed to a rawmaterial which is less expensive than glucose, the cost for producing ahighly unsaturated fatty acid-containing lipid can be reduced. When astarch which is a raw material for glucose is used as a medium carbonsource, the raw material cost can be reduced, however, α-glucosidaseproduced by a Mortierella microorganism has the low activity ofdegrading a starch as described above and, therefore, the starch can notbe sufficiently assimilated by said microorganism. In addition, aculture medium containing glucose at a high concentration has a highosmotic pressure, and this becomes a cause for delay of the growth of aMortierella microorganism, reduction in its productivity of a highlyunsaturated fatty acid-containing lipid and change in the morphologicalform. Therefore, it is difficult to produce a highly unsaturated fattyacid-containing lipid at a low cost by a batch culture method in whichglucose is not sequentially added during culturing and glucose iscontained at a high concentration at the time of the initiation ofculturing. As a means to solve this problem, a feeding culture method inwhich glucose is sequentially added to the culture medium is used.However, since the feeding culture not only requires an additionalapparatus for sequentially adding glucose but also increases the numberof works and working time in the culturing step, this method increasesthe cost for producing a highly unsaturated fatty acid-containing lipid.In addition, although glucose is produced by enzymatic degradation ofstarch with various amylases, it is necessary to isolate and purifyglucose after the enzymatic treatment. Thus, the cost for enzymatictreatment and isolation and purification makes glucose more expensivethan starch.

Production of a highly unsaturated fatty acid-containing lipid utilizinga starch or a soluble starch as a medium carbon source has been reported(Shinmen et al., Appl. Microbiol. Biotechnol. 31:11-16(1989), Aki etal., JAOCS 78:599-604 (2001)). However, the yield of a highlyunsaturated fatty acid-containing lipid per sugar in said culture mediumis lower than that of the case where the same amount of glucose is usedas a carbon source due to the incomplete assimilation of a starch or asoluble starch.

DISCLOSURE OF THE INVENTION

The present invention provides a process for producing a highlyunsaturated fatty acid-containing lipid at a lower cost than existingones, comprising the use of a medium carbon source, which is lessexpensive than glucose, does not participate in increasing osmoticpressure of the culture medium and can be assimilated by a Mortierellamicroorganism.

The α-glucosidase produced by a Mortierella microorganism has a lowactivity of degrading a starch as described above. In order to use astarch as a medium carbon source, it is necessary to degrade the starchto a saccharified starch which can be easily degraded by α-glucosidaseproduced by a Mortierella microorganism. However, when a starch iscompletely degraded to glucose, an increase in osmotic pressure due toan increase of monosaccharide is caused, having various negativeinfluences on the production of a highly unsaturated fattyacid-containing lipid as described above. Then, the present inventorscame up with an idea of controlling the average chain length of anoligoglucose in a saccharified starch at a proper length which can beeasily degraded by α-glucosidase produced by a Mortierellamicroorganism, by selecting a sugar degrading enzyme forsaccharification of starch and conditions of saccharification with theenzyme. The present inventors compared the productivity of a highlyunsaturated fatty acid-containing lipid, which was extracted from thecells obtained by culturing a Mortierella microorganism using, as amedium carbon source, a saccharified starch prepared by saccharificationof a starch using various sugar degrading enzymes under a variety ofconditions, with the productivity of the case where culturing isperformed by a batch culture using the same amount of starch or glucoseas a medium carbon source and with the productivity of the case whereculturing is performed by a feeding culture by adding the same amount ofglucose as a medium carbon source. As a result, the present inventorsfound that, when a saccharified starch is used as a carbon source, ahigher productivity is exhibited as compared to the case of the batchculture using a starch or glucose as a medium carbon source, and thatthe productivity was equivalent to that of the feeding culture usingglucose as a medium carbon source. The present inventors conductedfurther studies and finally completed the present invention.

That is, the present invention relates to:

(1) a process for producing a highly unsaturated fatty acid-containinglipid, which comprises culturing a microorganism belonging to the genusMortierella with the use of a culture medium containing a saccharifiedstarch, and collecting a highly unsaturated fatty acid-containing lipidfrom the culture,

(2) the process according to the above (1), wherein the saccharifiedstarch is obtained by treating a starch or a soluble starch with asaccharifying enzyme;

(3) a process for producing a highly unsaturated fatty acid-containinglipid, which comprises culturing a microorganism belonging to the genusMortierella with the use of a culture medium containing (a) a starch ora soluble starch and (b) a saccharifying enzyme, and collecting a highlyunsaturated fatty acid-containing lipid from the culture;

(4) a process for producing a highly unsaturated fatty acid-containinglipid, which comprises mixed-culturing a Mortierella microorganism and amicroorganism producing a saccharifying enzyme with the use of a culturemedium containing a starch or a soluble starch, and collecting a highlyunsaturated fatty acid-containing lipid from the culture;

(5) the process according to the above (4), wherein the microorganismproducing a saccharifying enzyme is a microorganism belonging to thegenus Aspergillus;

(6) the process according to the above (5), wherein the microorganismbelonging to the genus Aspergillus is Aspergillus oryzae or Aspergilluskawachii;

(7) the process according to any one of the above (1) to (6), whereinthe microorganism belonging to the genus Mortierella is a microorganismbelonging to the subgenus Mortierella;

(8) the process according to the above (7), wherein the microorganismbelonging to the subgenus Mortierella is Mortierella alpina orMortierella alliacea; and

(9) the process according to any one of the above (1) to (8), whereinthe highly unsaturated fatty acid is one or more fatty acid(s) selectedfrom the group consisting of γ-linolenic acid, dihomo-γ-linolenic acid,arachidonic acid, eicosapentaenoic acid, mead acid, 6,9-octadecadienoicacid and 8,11-eicosadienoic acid.

BEST MODE FOR CARRYING OUT THE INVENTION

A microorganism used in the present invention may be any microorganismbelonging to the genus Mortierella which can produce a lipid containinghighly unsaturated fatty acids. As such a microorganism, bacteriumstrains described, for example, in MYCOTAXON, Vol.XLIV, No. 2,pp.257-265 (1992) can be used, and specific examples thereof includemicroorganisms belonging to the subgenus Mortierella such as Mortierellaelongata IF08570, Mortierella exigua IF08571, Mortierella hygrophilaIF05941, Mortierella alpina IF08568, ATCC16266, ATCC32221, ATCC42430,CBS 219.35, CBS224.37, CBS250.53, CBS343.66, CBS527.72, CBS528.72,CBS529.72, CBS608.70, CBS754.68, etc. and microorganisms belonging tothe subgenus Micromucor such as Mortierella isabellina CBS194.28,IF06336, IF07824, IF07873, IF07874, IF08286, IF08308, IF07884,Mortierella nana IF08190, Mortierella ramanniana IF05426, IF08186,CBS112.08, CBS212.72, IF07825, IF08184, IF08185, IF08287, Mortierellavinacea CBS236.82, etc.

Any of those strains can be obtained from Institute for Fermentation,Osaka City, and American Type Culture Collection (ATCC) in USA, andCentraalbureau voor Schimmelcultures (CBS) in Netherlands without anyrestriction. Alternatively, a strain Mortierella elongata SAM0219(National Institute of Bioscience and Human-Technology, depositionNo.8703) (National Institute of Bioscience and Human-Technology,conditional deposition No.1289) which was isolated from a soil by thepresent inventors may be used. Any strain belonging to those typecultures or strains isolated from the natural world can be used as theyare. Also, as the microorganism used in the present invention, a mutantor a genetically recombinant strain of a Mortierella microorganism (wildstrain) may be used. Among them, preferable examples include a mutantand a genetically modified strain in which, when cultured under the sameculturing conditions as those of a wild strain, the amount of particularor all highly unsaturated fatty acid(s) in a lipid is larger, or a totallipid amount is larger, as compared to that of a highly unsaturatedfatty acid lipid produced by the original wild strain, or those whichcan attain both of them. Examples of a mutant which contains largeramount of a particular highly unsaturated fatty acid in a lipid includeMortierella alpina SAM1861 which lacks the Δ12 desaturation enzymeactivity (National Institute of Bioscience and Human-Technology,conditional deposition No. 3590, FERM BP-3590), and Mortierella alpineSAM1860 which lacks the Δ5 desaturation enzyme activity (NationalInstitute of Bioscience and Human-Technology, conditional deposition No.3589, FERMBP-3589). Alternatively, Mortierella SAM2197having resistanceto high concentration of glucose (FERM BP-6261) may be used.

Examples of a process for producing a highly unsaturated fattyacid-containing lipid of the present invention include the threeembodiments of (i) culturing the aforementioned Mortierellamicroorganism with the use of a culture medium containing a saccharifiedstarch, and collecting a highly unsaturated fatty acid-containing lipidfrom the culture, (ii) culturing the aforementioned Mortierellamicroorganism with the use of a culture medium containing (a) a starchor a soluble starch and (b) a saccharifying enzyme, and collecting ahighly unsaturated fatty acid-containing lipid from the culture, or(iii) mixed-culturing the aforementioned Mortierella microorganism and amicroorganism producing a saccharifying enzyme (hereinafter, referred toas saccharifying enzyme-producing microorganism) with the use of aculture medium containing a starch or a soluble starch, and collecting ahighly unsaturated fatty acid-containing lipid from the culture. Eachembodiment will be separately explained below.

As a first embodiment of the present invention, a process for producinga highly unsaturated fatty acid-containing lipid, which comprisesculturing the aforementioned Mortierella microorganism with the use of aculture medium containing a saccharified starch, and collecting a highlyunsaturated fatty acid-containing lipid from the culture will bedescribed. As mentioned above, the use of a saccharified starch which isobtained by saccharification of a starch in advance as a carbon sourceof the culture medium, has advantages that said saccharified starch iseasily assimilated by a Mortierella microorganism and an increase ofosmotic pressure in the culture medium is suppressed, whereby the yieldof a highly unsaturated fatty acid-containing lipid per carbon sourcecan be increased.

Culturing of a single Mortierella microorganism in the presentembodiment may be performed according to the conventional method, exceptthat a saccharified starch is used as a carbon source of the culturemedium. For example, there can be exemplified a method in which a liquidculture medium or a solid culture medium is inoculated with a spore or ahypha of the Mortierella microorganism or a culture solution obtained bypre-culture, followed by culturing.

A culture medium for culturing a microorganism in the said embodiment isnot particularly limited as far as the culture medium contains asaccharified starch as a medium carbon source. Inter alia, it isdesirable that the saccharified starch used as a medium carbon sourcehas the saccharification degree at which it can be most easily degradedby α-glucosidase produced by the Mortierella microorganism which is tobe cultured, that is, an average chain length of oligoglucose in asaccharified starch. Specifically, in view of an increase in osmoticpressure and a chain length of oligoglucose in the saccharified starchwhich can be assimilated by a Mortierella microorganism, it ispreferable to use the saccharified starch of which reducing sugar/totalsugar ratio, i.e., a saccharification degree, is about 2 to 90%,preferably about 30 to 90%.

A saccharified starch can be obtained by treating a starch or a solublestarch with a saccharifying enzyme and, by further selecting thesaccharifying enzyme and setting the conditions of saccharification withthe enzyme, the saccharified starch having the above desiredsaccharification degree can be obtained. A saccharifying enzyme is notparticularly limited as far as it is a sugar degrading enzyme which candegrade starch, etc., but an endo-type amylase such as α-amylase havingrandom degradation patterns, or an exo-type amylase such as β-amylasewhich cleaves maltose units from an end of a starch molecule ispreferable. Alternatively, saccharifying enzymes having differentactions of mechanism may be used by combining thereof. A saccharifyingenzyme used in saccharification can be obtained by culturing amicroorganism producing the saccharifying enzyme, and obtaining theenzyme from the culture by the known method. Alternatively, acommercially available saccharifying enzyme may be used. Examples ofcommercially available saccharifying enzymes include an α-amylase suchas amylase AD “Amano”1 (manufactured by Amano Enzyme Inc.) and apullulanase such as Pullulanase “Amano”3 (manufactured by Amano EnzymeInc.). Also, the saccharified starch can be obtained by other knownmethods such as degradation of starch or soluble starch with an acid. Asan acid degradation method to degrade a starch or a soluble starch withan acid to obtain a saccharified starch, a preferable example is atreatment using oxalic acid. Further, as the saccharified starch, acommercially available saccharified starch may be used. Examples ofcommercially available saccharified starches include Fujisyrup C-75S,Fujisyrup C-75, HMTP-75 and A-75 (all of them are manufactured by KatoKagaku). Since those commercially available saccharified starches havedifferent saccharification degrees, a saccharified starch which issuitable for carrying out the present invention can be selected.

A culture medium for culturing a microorganism used in the presentembodiment may include, as an auxiliary raw material for compensatingthe saccharified starch, a common carbon source of a culture medium,such as glucose, fructose, xylose, saccharose, maltose, molasses,glycerol, mannitol, citric acid, etc. In addition, as a nitrogen sourceof the culture medium, an organic nitrogen source such as soybeanpowder, soybean flake, defatted soybean powder, edible soybean protein,soybean peptide, soybean flour, peptone, yeast extract, malt extract,meat extract, casamino acid, corn steep liquor, urea, etc., or aninorganic nitrogen source such as ammonium nitrate, ammonium sulfate,etc. may be contained in the culture medium for culturing amicroorganism used in the present embodiment. Among them, a preferableexample of the medium nitrogen source includes heat denatured defattedsoybean and, in particular, those which have been heat-denatured atabout 70 to 90° C. and from which an ethanol-soluble component wasremoved, are more preferable. Further, a culture medium for culturing amicroorganism used in the present embodiment may contain, as amicronutrient source, inorganic salts comprising phosphoric acid,potassium, sodium, magnesium and/or calcium, metal ions such as iron,copper, zinc, manganese, nickel, cobalt, etc. and vitamins. Interalia,it is preferable that one or more inorganic salt(s) selected from thegroup consisting of phosphoric acid, potassium, sodium, magnesium andcalcium is/are contained.

In addition, in order to increase the yield of highly unsaturated fattyacids, a culture medium for culturing a microorganism used in thepresent embodiment may contain, as a precursor of the highly unsaturatedfatty acids, for example, hydrocarbons such as hexadecane andoctadecane; fatty acids such as oleic acid and linolenic acid or a saltthereof; triacyl glycerol and fatty acid esters such as fatty acid ethylester, glycerin fatty acid ester, sorbitan fatty acid ester, etc.; fatsor oils such as olive oil, soybean oil, rapeseed oil, cotton seed oiland palm oil. One kind only of the aforementioned compounds may becontained alone, or a combination of two or more of them may becontained.

The aforementioned carbon source, nitrogen source and other mediumcomponent may be added to a culture medium before initiation ofculturing and/or to a culture medium during culturing. These mediumcomponents may be added at once, or may be added sequentially or bydividing into plural times with the lapse of time. These mediumcomponents may be added alone or by mixing them in advance, aftersterilization. A method of sterilization, and an order of addition arenot particularly limited. Preferably, it is desirable that a carbonsource and a nitrogen source are separately sterilized, and it isdesirable that salts are added before completion of the logarithmicgrowth, more preferably before the intermediate stage of the logarithmicgrowth. Regarding other medium components which have no influence on theconcentration of phosphoric acid ion, potassium ion, sodium ion,magnesium ion and calcium ion, the time of addition is not particularlylimited as far as those components have such a concentration that growthof a Mortierella microorganism is not inhibited.

Practically, it is desirable that a total amount of a carbon source tobe added is generally about 0.1 to 40% by weight, preferably about 1 to25% by weight, and a total amount of a nitrogen source to be added isabout 0.01 to 10% by weight, preferably about 0.1 to 10% by weight. Morepreferably, a carbon source and a nitrogen source may be added duringculturing, with the amount of initially added carbon source being 1 to12% by weight and the amount of initially added nitrogen source beingabout 0.1 to 8% by weight. In addition, the amount of the aforementionedprecursor of highly unsaturated fatty acids to be added is about 0.001to 10%, preferably about 0.5 to 10% relative to the culture medium.

Culturing conditions are not particularly limited, and may be accordingto the conventional method. For example, a culturing temperature isabout 5 to 40° C., preferably about 20 to 30° C. Alternatively, after aMortierella microorganism is proliferated at about 20 to 30° C.,culturing may be continued at about 5 to 20° C. to produce a highlyunsaturated fatty acid. The pH of the culture medium is about 4 to 10,preferably about 5 to 8. Examples of the culturing method includeaerated culture underagitation, shakeculture, static culture, etc.Culturing is usually performed for about 2 to 20 days. By culturing asmentioned above, a highly unsaturated fatty acid-containing lipid isproduced and accumulated in the Mortierella microorganism. In thepresent invention, aerated culture under agitation using a liquid mediumis preferable.

Next, the highly unsaturated fatty acid-containing lipid is collectedfrom the thus obtained culture. Examples of said “culture” include aculture solution during the production of a lipid by culturing and thesterilized product thereof, or a culturing solution after completion ofculturing and the sterilized product thereof, or cultured cellscollected from each of them and the dried products thereof. As a methodof collecting a highly unsaturated fatty acid-containing lipid from theaforementioned culture, the known method may be used. For example, anobjective lipid may be collected from cultured cells by the followingmethod.

After completion of culturing, cultured cells are obtained from theculture solution by a conventional solid-liquid separation method suchas centrifugation and/or filtration. The cultured cells are preferablywashed with water, ground and dried. Drying may be performed bylyophilization, air drying, etc. The dried cells are preferably treatedby extraction with an organic solvent under a nitrogen stream. Examplesof the organic solvent used include ether, hexane, methanol, ethanol,chloroform, dichloromethane, petroleum ether, etc. Alternatively, betterresult can be also obtained by an alternate extraction with methanol andpetroleum ether, or an extraction using a solvent of one layer system ofchloroform-methanol-water. Inter alia, it is preferable to performextraction using hexane. By distilling an organic solvent off from theextract under reduced pressure, a lipid containing a highly unsaturatedfatty acid of high concentration can be obtained. Alternatively,extraction may be performed using wet cells, in place of theaforementioned method. In this case, a solvent which is compatible withwater such as methanol, ethanol, etc., or a mixed solvent compatiblewith water which contains the aforementioned solvent and water and/orother solvent is used. Other procedures are the same as described above.

As a second embodiment of the present invention, a process for producinga highly unsaturated fatty acid-containing lipid, which comprisesculturing the aforementioned Mortierella microorganism using a culturemedium containing (a) a starch or a soluble starch and (b) asaccharifying enzyme, and collecting a highly unsaturated fattyacid-containing lipid from the culture will be described. In the presentembodiment, a saccharified starch produced from a starch or a solublestarch contained in the culture medium with a saccharifying enzyme isused as a medium carbon source. That is, in the present embodiment,saccharification of starch and culturing of a Mortierella microorganismare performed at the same time. More specifically, the process is suchthat a saccharifying enzyme is added to a culture medium containing astarch or a soluble starch as a carbon source to convert the starch orthe soluble starch into a saccharified starch which is easily degradedby α-glycosidase produced by a Mortierella microorganism, and glucosewhich is produced from the saccharified starch by the α-glycosidase isassimilated by the Mortierella microorganism to produce a highlyunsaturated fatty acid-containing lipid.

A starch used in the present embodiment is not particularly limited, butspecific examples include rice starch, cane starch, potato starch,tapioca starch, wheat starch and corn starch, and the processed starchand α-starch thereof. Among starches, those which are soluble in hotwater are called soluble starch.

As a saccharifying enzyme used in the present embodiment, anysaccharifying enzyme may be used as far as it is an enzyme used forsaccharification. Specifically, a saccharifying enzyme prepared from asaccharifying enzyme-producing microorganism and a commerciallyavailable saccharifying enzyme can be used as mentioned in the firstembodiment. Examples of the saccharifying enzyme used in the presentembodiment include, as in the first embodiment, an endo-type amylasesuch as α-amylase having random degradation patterns, and a β-amylasewhich cleaves maltose units from an end of a starch molecule arepreferable, and also an exo-type amylase such as glucoamylase whichsequentially degrades starch from a non-reducing end to produce glucosemay be used. This is because, since glucose produced by glucoamylase isimmediately assimilated by a Mortierella microorganism, adverseinfluences due to an increase in osmotic pressure of a culture mediumare not caused.

The present embodiment is entirely the same as the first embodimentexcept that (a) a starch or a soluble starch and (b) a saccharifyingenzyme are used as a medium component for culturing microorganism inplace of the saccharified starch. The medium component for culturingmicroorganism in the present embodiment may contain the aforementionedsaccharified starch.

Next, as a third embodiment of the present invention, a process forproducing a highly unsaturated fatty acid-containing lipid, whichcomprises mixed-culturing the aforementioned Mortierella microorganismand saccharifying enzyme-producing microorganism with the use of aculture medium containing a starch or a soluble starch, and collecting ahighly unsaturated fatty acid-containing lipid from the culture will bedescribed. In the present embodiment, a saccharified starch produced bya saccharifying enzyme produced by a saccharifying enzyme-producingmicroorganism from a starch or a soluble starch contained in a culturemedium is used as a medium carbon source. That is, the presentembodiment is a process in which a saccharified starch is produced bymixed-culturing a saccharifying enzyme-producing microorganism used inthe saccharification together with a Mortierella microorganism in aculture medium containing a starch or a soluble starch as a mediumcarbon source, and the Mortierella microorganism produces a highlyunsaturated fatty acid-containing lipid from the saccharified starch.

In the present embodiment, a Mortierella microorganism is cultured as amain microorganism, and a saccharifying enzyme-producing microorganismis cultured as an auxiliary microorganism. As the saccharifyingenzyme-producing microorganism, any microorganisms can be used as far asthey are a microorganism which secretes a saccharifying enzyme, andexamples thereof include filamentous fungus belonging to the genusAspergillus, Rhizopus and Trichoderma, and bacteria belonging toBacillus, Microbacterium and Klebsiella. Among them, as thesaccharifying enzyme-producing microorganism, a fungus belonging to thegenus Aspergillus which produces α-amylase is preferable and, inparticular, Aspergillus oryzae and Aspergillus kawachii are morepreferable. Also, a microorganism secreting the glucoamylase can bepreferably used as an auxiliary microorganism. Examples of theglucoamylase-secreting microorganism include Aspergillus microorganismsand Rhizopus microorganisms. In addition, by simple-culturing a mutantor a genetically recombinant strain of a Mortierella microorganism whichis imparted with the saccharifying enzyme-producing ability, the sameeffect as that of mixed-culturing with a saccharifying enzyme-producingmicroorganism can be obtained.

The present embodiment is entirely the same as the first embodiment,expect that a starch or a soluble starch is used as a medium componentfor culturing microorganism in place of the saccharified starch, and aMortierella microorganism and a saccharifying enzyme-producingmicroorganism are mixed and cultured in place of the simple-culturing ofa Mortierella microorganism. In addition, the medium component forculturing microorganism in the present embodiment may contain theaforementioned saccharified starch. In this case, it is preferable thata saccharification degree of the saccharified starch, that is, a ratioof reducing sugar/total sugar is about 0 to 80%, preferably about 0 to70%. When a Mortierella microorganism and a succharifyingenzyme-produding microorganism are mixed-cultured, both microorganismsmay be cultured at the same time, or after one of the microorganisms iscultured to some extent, the other microorganism may be added to performmixed-culturing.

A highly unsaturated fatty acid-containing lipid obtained by the abovemethod can be used in various applications such as animal feeds andfoods. It is preferable that the lipid contains one or more highlyunsaturated fatty acid (s) selected from the group consisting ofγ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,eicosapentaenoic acid, mead acid, 6,9-octadecadienoic acid and8,11-eicosadienoic acid. Separation and purification of such highlyunsaturated fatty acid can be performed by solvent extraction and, afterdesolvation, by deacidification, decolorization, deodorization, gumremoval or cooling separation according to the conventional methods.Preferable examples include the method described in InternationalPublication WO98/39468.

EXAMPLES

The invention will be described in detail below by way of Examples, butit goes without saying that the present invention is not limited tothem. A component composition in a culture medium is shown by % byweight in all cases.

Example 1

As an arachidonic acid-producing microorganism, Mortierella SAM2197(FERM BP-6261) was used.

Each 5 L of the culture medium 1-A, 1-B and 1-C shown in Table 1 wasprepared in a culturing tank with 10 L capacity, and inoculated with thepre-cultured solution prepared in advance by flask culturing, followedby culturing. The amylase-treated starch for a culture medium 1-A usedherein was the starch obtained by adding α-amylase (manufactured by WakoPure Chemical Industries, Ltd., code No.011-16881) to a starch andincubating at 37° C. for 30 minutes. A reducing sugar was measured by aSomogyi-Nelson method, and a total sugar was measured by a Phenol-H₂SO₄method. As a result of the measurement, the reducing sugar/total sugarratio of the amylase-treated starch for the culture medium 1-A was 38%,and the reducing sugar/total sugar ratio of the soluble starch for theculture medium 1-B was 0%.

After aerated culture under agitation for 7 days, the amount of theproduced arachidonic acid of 4.2 g/L was obtained in the culture medium1-A, 1.2 g/L in the culture medium 1-B, and 4.3 g/L in the culturemedium 1-C. An amount of produced arachidonic acid was calculated asfollows. That is, a culturing solution was filtered through filter paperto recover a cell cake, and cells were dried at 105° C. and weighed. Avalue of the weight after drying was divided by value of the volume ofthe culturing solution which was subjected to filtration, to obtain adried cell concentration. About 20 mg of the dried cell piece wasprecisely weighed into a screw-capped test tube, to this were added 2 mLof hydrochloric acid-methanol solution and 1 mL of dichloromethane, andreacted at 50° C. for 3 hours. After the reaction, hexane was added toextract fatty acid methyl ester, and the recovered hexane layer wasconcentrated under reduced pressure. The resulting fatty acid methylester was dissolved in a prescribed amount of acetonitrile, fractionatedby gas chromatography, and each fatty acid methyl ester was quantitatedfrom the peak area. The result of quantitation was divided by theprecisely weighed value of cell piece to obtain each fatty acid contentper dried cell. The resulting each fatty acid content was multiplied bythe dried cell concentration to obtain the produced amount of each fattyacid.

TABLE 1 Culture medium No. 1-A 1-B 1-C Amylase-treated starch  12%   0%  0% Soluble starch   0%  12%   0% Glucose   0%   0%  12% Soybean powder  3%   3%   3% Soybean oil 0.2% 0.2% 0.2% Anti-foaming agent 0.1% 0.1%0.1% Produced amount of 4.2 g/L 1.2 g/L 4.3 g/L arachidonic acid

As apparent from the above table, an amount of produced arachidonic acidwas small in a culture medium using a soluble starch, however, by usinga saccharified starch obtained by treatment of starch with amylase, ahigh produced amount of arachidonic acid, which is equivalent to that ofa medium using glucose, was obtained.

Example 2

As an arachidonic acid-producing microorganism, Mortierella SAM2197(FERM BP-6261) was used. A culture medium (50 mL) containing 24% ofsoluble starch and 1.5% of yeast extract was prepared in a 500 mL flask.The culture medium (50 mL) was inoculated with 1×10⁵ spores ofMortierella SAM2197, inoculated with each of various amounts of a sporesuspension of Aspergillus oryzae, and cultured for 7 days under thecondition of 28° C. and at 160 rpm. As a control, a culture mediumcontaining 12% of glucose and 1.5% of yeast extract was prepared asdescribed above, and inoculated with Mortireilla SAM2197 (FERMBP-6261),followed by culturing. On the 5^(th) day of culturing, 12% of glucosewas added, followed by culturing for 7 days under the condition of 28°C. and 160 rpm. As a result of culturing, as shown in Table 2, theamount of produced arachidonic acid was 0.56 g/L at maximum when aninoculation amount of an Aspergillus oryzae spore suspension was 0.1 mL,which is nearly 2-fold that of the case where a soluble starch was usedin single culturing of Mortierella (0.3 g/L), and is a production amountequivalent to the case where glucose was used as a carbon source bysingle culturing. When the amount of Aspergillus spore inoculation was 1mL, the amount of produced arachidonic acid was reduced, which suggeststhat the growth of Mortierella may be suppressed in some cases due tothe influence of growth of Aspergillus. Thus, it has been proved that,in the case of mixed-culturing of a Mortierella microorganism and asaccharifying enzyme-producing microorganism, it is preferable toculture with the former being a main microorganism.

TABLE 2 Inoculation amount of Aspergillus spore Amount of producedCarbon source suspension arachidonic acid Soluble starch   0 mL 0.30 g/LSoluble starch 0.01 mL 0.50 g/L Soluble starch  0.1 mL 0.56 g/L Solublestarch   1 mL 0.16 g/L Glucose   0 mL 0.58 g/L

Example 3

As an arachidonic acid-producing microorganism, Mortierella alpineCBS754.68 was used. A culture medium (50 mL) containing each 4.5% ofvarious sugars shown in Table 3, and 1% of yeast extract was prepared ina 500 mL flask, and inoculated with 1×10³ spores, followed by culturing.Culturing was performed for 5 days under the condition of 28° C. and 100rpm. A method for saccharification of the starch (2), (7) and (8) wasconducted in such a manner that an α-amylase and a pullulanase wereadded to a starch, and incubated at 37° C. for 30 minutes. As α-amylase,amylase AD “Amano”1 (manufactured by Amano Enzyme Inc.) was used and, aspullulanase, pullulanase “Amano”3 (manufactured by Amano Enzyme Inc.)was used.

As a result of culturing, amounts of produced arachidonic acid as shownin Table 3 were obtained, and it was shown that the preferable range ofsaccharification degree as described above exists.

TABLE 3 Reducing sugar/ Produced amount of Carbon source total sugarratio arachidonic acid Glucose 100% 0.65 g/L Soluble starch 0% 0.11 g/L(1) Saccharified 18% 0.43 g/L starch (2) Saccharified 27% 0.58 g/Lstarch (3) Saccharified 33% 0.61 g/L starch (4) Saccharified 50% 0.61g/L starch (5) Saccharified 55% 0.69 g/L starch (6) Saccharified 80%0.69 g/L starch (7) Saccharified 92% 0.65 g/L starch (8) Details ofcarbon source (1) Stabilose K, manufactured by Matsutani ChemicalIndustry Co., Ltd. (2) Starch saccharified with α-amylase andpullulanase (3) Fujisyrup C-75S, manufactured by Kato Kagaku (4)Fujisyrup C-75, manufactured by Kato Kagaku (5) HMTP-75 (M-70),manufactured by Kato Kagaku (6) A-75, manufactured by Kato Kagaku (7)Starch saccharified with α-amylase and pullulanase (8) Starchsaccharified with α-amylase and pullulanase

Example 4

As an arachidonic acid-producing microorganism, Mortierella alpinaCBS754.68 was used. Seed culturing was performed using a culture mediumcontaining yeast extract and glucose as a nutrient source, andinoculated into a culture medium which was prepared in a 50 L culturingtank for fermentation. A medium composition of the regular culturing wasany of three sugar compositions of 4-A, 4-B and 4-C shown in Table 4,and the common medium composition other than sugar was soybean powder4%, soybean oil 0.1%, KH₂PO₄ 0.3%, Na₂SO₄ 0.1%, CaCl₂.2H₂O 0.05% andMgCl₂.6H₂O 0.05%. The reducing sugar/total sugar ratio of thesaccharified starch used was 30%.

As a result of culturing for 10 days, the amounts of producedarachidonic acid in the culture mediums 4-A, 4-B and 4-C were 13.5 g/L,7 g/L and 13.3 g/L, respectively. As elucidated by this result, by usinga saccharified starch, not only avoidance of growth inhibition due tohigh concentration of glucose, but also and reduction in the cost forculturing due to fewer feedings can be achieved.

TABLE 4 Culture medium No. 4-A 4-B 4-C Carbon source SaccharifiedGlucose Glucose starch Initial sugar 1.8% 6% 6% concentration Flowingsugar concentration 1 day flowing 4.1% 0% 0% 2 day flowing 4.1% 6% 6% 3day flowing 3.6% 6% 6% 4 day flowing 2.7% 0% 0% 5 day flowing 1.7% 0% 0%Total carbon source  18% 18% 18% Amount of produced 13.5 g/L 7.0 g/L13.3 g/L arachidonic acid

Example 5

As a mead acid-producing microorganism, Mortierella alpine SAM1861(National Institute of Bioscience and Human-Technology, Conditionaldeposition No. 3590, FERM BP-3590) was used and, as dihomo-γ-linolenicacid-producing microorganism, Mortierella alpine SAM1860 (NationalInstitute of Bioscience and Human-Technology, conditional deposition No.3589, FERM BP-3589) was used. Each of three kinds of culture mediums (50mL) shown in Table 5 was prepared in a 500 mL flask, and inoculated with1×10³ spores to initiate culturing. Culturing was performed for 7 daysunder the condition of 24° C. and 100 rpm.

TABLE 5 Culture medium No. 5-A 5-B 5-C Saccharified starch (*)   5%   0%  0% Soluble starch   0%   5%   0% Glucose   0%   0%   5% Soybean powder1.5% 1.5% 1.5% KH₂PO₄ 0.2% 0.2% 0.2% Anti-foaming agent 0.1% 0.1% 0.1%Strain: SAM1861 0.44 g/L 0.12 g/L 0.45 g/L Amount of produced mead acidStrain: SAM1860 0.55 g/L 0.20 g/L 0.60 g/L Amount of produceddihomo-γ-linolenic acid (*) Saccharified starch having the reducingsugar/total sugar ratio = 35% was used.

In the case where a soluble starch is used for culturing, both of theamounts of produced mead acid and produced dihomo-γ-linolenic acid werelow, however, by using a saccharified starch, a large production amountequivalent to that of a glucose culture medium was obtained.

INDUSTRIAL APPLICABILITY

According to the present invention, a starch which is less expensivethan glucose conventionally used as a medium carbon source is used forculturing a Mortierella microorganism, thus the cost for raw materialsof a culture medium can be reduced and, consequently, the cost forproducing a highly unsaturated fatty acid-containing lipid can bereduced. In addition, by using a saccharified starch as a medium carbonsource, an increase of osmotic pressure in the culture medium can besuppressed, resulting in an increase in the yield of a highlyunsaturated fatty acid-containing lipid per carbon source. Since anincrease of osmotic pressure can be suppressed as mentioned above, inthe present invention, unlike the case using glucose as a medium carbonsource, feeding culture is not necessarily conducted. Therefore,facilities, the number of works and working time in the culturing stepcan be saved and, consequently, the cost for producing a highlyunsaturated fatty acid-containing lipid can be reduced.

The invention claimed is:
 1. A process for producing a highlyunsaturated fatty acid-containing lipid, which comprises culturing amicroorganism belonging to the genus Mortierella subgenus Mortierellawith the use of a culture medium containing a saccharified starch, andcollecting a highly unsaturated fatty acid-containing lipid from theculture, wherein said saccharified starch has a saccharification degreeof 30% to 92% and has productivity of a highly unsaturatedfatty-acid-containing lipid comparable to that of glucose as a mediumcarbon source.
 2. The process according to claim 1, wherein thesaccharified starch is obtained by treating a starch or a soluble starchwith a saccharifying enzyme.
 3. A process for producing a highlyunsaturated fatty acid-containing lipid, which comprises culturing amicroorganism belonging to the genus Mortierella subgenus Mortierellawith the use of a culture medium containing (a) a starch or a solublestarch and (b) a saccharifying enzyme, and collecting a highlyunsaturated fatty acid-containing lipid from the culture, wherein asaccharified starch is produced from said starch or soluble starchcontained in the culture medium with said saccharifying enzyme, and saidsaccharified starch has a saccharification degree of 30% to 92%, and hasproductivity of a highly unsaturated fatty-acid containing lipidcomparable to that of glucose as a medium carbon source and wherein saidsaccharified starch is used as a medium carbon source.
 4. A process forproducing a highly unsaturated fatty acid-containing lipid, whichcomprises mixed-culturing a Mortierella microorganism and amicroorganism producing a saccharifying enzyme with the use of a culturemedium containing a starch or a soluble starch, and collecting a highlyunsaturated fatty acid-containing lipid from the culture; wherein saidMortierella microorganism is of genus Mortierella subgenus Mortierella;wherein said microorganism producing a saccharifying enzyme isAspergillus oryzae or Aspergillus kawachii; wherein said Mortierellamicroorganism is cultured as a main microorganism and said saccharifyingenzyme-producing microorganism is cultured as an auxiliarymicroorganism, and wherein a saccharified starch is produced by saidsaccharifying enzyme from said starch or said soluble starch and saidsaccharified starch has a saccharification degree of 30% to 92% and isused as a medium carbon source and wherein said saccharified starch hasproductivity of a highly unsaturated fatty-acid containing lipidcomparable to that of glucose as a medium carbon source.
 5. The processaccording to claim 1, wherein the microorganism belonging to thesubgenus Mortierella is Mortierella alpina or Mortierella alliacea. 6.The process according to claim 1, wherein the highly unsaturated fattyacid is one or more fatty acid(s) selected from the group consisting ofγ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid,eicosapentaenoic acid, mead acid, 6,9-octadecadienoic acid and8,11-eicosadienoic acid.
 7. The process according to claim 1, whereinsaid saccharified starch has a saccharification degree of 33% to 92%. 8.A process for producing a highly unsaturated fatty acid-containinglipid, which comprises culturing a microorganism belonging to the genusMortierella subgenus Mortierella with the use of a culture mediumcontaining a saccharified starch, and collecting a highly unsaturatedfatty acid-containing lipid from the culture, wherein said saccharifiedstarch has a saccharification degree of 30 to 92% and has productivityof a highly unsaturated fatty-acid-containing lipid comparable to thatof glucose as a medium carbon source, and wherein the highly unsaturatedfatty acid is one or more fatty acid(s) selected from the groupconsisting of arachidonic acid, γ-linolenic acid, dihomo-γ-linolenicacid, eicosapentaenoic acid, mead acid, 6,9-octadecadienoic acid and8,11-eicosadienoic acid.